PTLs 1 to 3 disclose a biosensor which utilizes an excellent molecular recognition function of a receptor. Such biosensor comprises an artificial lipid membrane having receptors and ion channels.
Examples of a conventional artificial lipid membrane forming method are (1) a bubble spraying method, (2) an attaching method, and (3) μTAS (Micro Total Analysis System) (see NPL 1, for example).
FIG. 20 shows a conventional artificial lipid membrane forming method according to the bubble spraying method. In FIG. 20, the inside of a container 10 is divided by a flat plate 11 made of resin, such as Teflon (trademark) or polystyrene, having a hydrophobic property. Spaces divided by the flat plate 11 are filled with an electrolytic solution 12. A lipid solution 14 containing lipid molecules and an organic solvent is applied with a pipette 15 to a minute hole 13 formed on the flat plate 11. The surplus organic solvent contained in the lipid solution 14 applied to the minute hole 13 gradually move along a peripheral edge of the minute hole 13 to be removed. The artificial lipid membrane is formed in about 30 minutes to 3 hours after the application.
Examples of the lipid are phosphatides, such as diphytanoyl phosphatidylcholine and glycerol monooleate. Examples of the organic solvent are saturated hydrocarbons, such as decane, hexadecane, and hexane.
Each of FIGS. 21(a) to 21(c) shows a conventional artificial lipid membrane forming method according to the attaching method. In FIG. 21(a), the inside of a container 20 is divided by a flat plate 21 having a hydrophobic surface. The flat plate 21 is made of resin, such as Teflon (trademark) or polystyrene.
First, as a pretreatment, squalene is applied to a minute hole 22 formed on the flat plate 21. An electrolytic solution 23 is added through an inlet 24 to one of chambers of the container 20 such that a solution level of the electrolytic solution 23 does not exceed the height of a lower end of the minute hole 22. Next, a lipid solution (mixture of lipid molecules 25 and an organic solvent) is dropped onto the electrolytic solution 23 from above the container 20, and this mixture is left for several minutes. As shown in FIG. 21(a), a lipid monolayer is formed on a gas-liquid interface of the electrolytic solution 23. The lipid molecule 25 has a hydrophilic portion and a hydrophobic portion, and the hydrophilic portion of the lipid molecule 25 is oriented toward the electrolytic solution 23.
Then, as shown in FIG. 21(b), the electrolytic solution 23 is added through the inlet 24 until the solution level of the electrolytic solution 23 exceeds the height of an upper end of the minute hole 22.
The same steps as above are carried out in the other chamber of the container 20. To be specific, as shown in FIG. 21(c), an electrolytic solution 26 is added through an inlet 27 such that the solution level of the electrolytic solution 26 does not exceed the height of the lower end of the minute hole 22. Next, the lipid solution is dropped onto the electrolytic solution 26 from above the container 20, and this mixture is left for several minutes. The lipid monolayer is formed on the gas-liquid interface of the electrolytic solution 26. The electrolytic solution 26 is added through the inlet 27 until the solution level of the electrolytic solution 26 exceeds the height of the upper end of the minute hole 22. Thus, this lipid monolayer formed later is attached to the lipid monolayer formed in advance at the minute hole 22. As a result, the artificial lipid membrane is formed at the minute hole 22.
It requires a high degree of skill to form the artificial lipid membrane stably and highly reproducibly by each of the above-described two methods.
In order to form further simple artificial lipid membranes, each of PTLs 1 to 4 discloses a method for forming the artificial lipid membrane using the μTAS technique.
FIG. 22 shows a compact artificial lipid membrane forming apparatus which is described in PTL 1 and uses the μTAS technique. The artificial lipid membrane forming apparatus shown in FIG. 22 comprises a first chamber 31 and a second chamber 33 which is isolated from the first chamber 31 by a dividing wall 32. The dividing wall 32 comprises at least one small hole 34 through which the first chamber 31 and the second chamber 33 are fluidically communicated with each other. The artificial lipid membrane is formed as below using the artificial lipid membrane forming apparatus. First, the first chamber 31 is filled with a first aqueous solution, and the second chamber 33 is then filled with a lipid solution. The first aqueous solution is brought in contact with the lipid solution through the small hole 34. Further, the lipid solution with which the second chamber 33 is filled is replaced with a second aqueous solution. Thus, an artificial lipid membrane 35 is formed at the small hole 34.